Differential regulation of synchronous versus asynchronous neurotransmitter release by the C2 domains of synaptotagmin 1

Synaptic vesicle fusion at many synapses has been kinetically separated into two distinct Ca2+-dependent temporal components consisting of a rapid synchronous phase followed by a slower asynchronous component. Mutations in the synaptic vesicle Ca2+ sensor Synaptotagmin 1 (Syt 1) reduce synchronous neurotransmission while enhancing the slower asynchronous phase of release. Syt 1 regulation of vesicle fusion requires interactions mediated by its tandem cytoplasmic C2 domains (C2A and C2B). Although Ca2+ binding by Syt 1 is predicted to drive synchronous release, it is unknown if Ca2+ interactions with either C2 domain is required for suppression of asynchronous release. To determine if Ca2+ binding by Syt 1 regulates these two phases of release independently, we performed electrophysiological analysis of transgenically expressed Syt 1 mutated at Ca2+ binding sites in C2A or C2B in the background of Drosophila Syt 1-null mutants. Transgenic animals expressing mutations that disrupt Ca2+ binding to C2A fully restored the synchronous phase of neurotransmitter release, whereas the asynchronous component was not suppressed. In contrast, rescue with Ca2+-binding mutants in C2B displayed little rescue of the synchronous release component, but reduced asynchronous release. These results suggest that the tandem C2 domains of Syt 1 play independent roles in neurotransmission, as Ca2+ binding to C2A suppresses asynchronous release, whereas Ca2+ binding to C2B mediates synchronous fusion.

[1]  J. Littleton,et al.  Synaptotagmins are trafficked to distinct subcellular domains including the postsynaptic compartment , 2004, The Journal of cell biology.

[2]  T. Südhof,et al.  Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse , 1994, Cell.

[3]  Edwin R. Chapman,et al.  Autapses and Networks of Hippocampal Neurons Exhibit Distinct Synaptic Transmission Phenotypes in the Absence of Synaptotagmin I , 2009, The Journal of Neuroscience.

[4]  B. Davletov,et al.  Mechanism of Calcium-independent Synaptotagmin Binding to Target SNAREs* , 2003, The Journal of Biological Chemistry.

[5]  J. Littleton,et al.  Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[6]  T. Südhof,et al.  Synaptotagmin-1, -2, and -9: Ca2+ Sensors for Fast Release that Specify Distinct Presynaptic Properties in Subsets of Neurons , 2007, Neuron.

[7]  K. White,et al.  Molecular analysis of the locus elav in Drosophila melanogaster: a gene whose embryonic expression is neural specific. , 1987, The EMBO journal.

[8]  E. Neher,et al.  Synaptotagmin Has an Essential Function in Synaptic Vesicle Positioning for Synchronous Release in Addition to Its Role as a Calcium Sensor , 2009, Neuron.

[9]  George J. Augustine,et al.  Synaptotagmin I Synchronizes Transmitter Release in Mouse Hippocampal Neurons , 2004, The Journal of Neuroscience.

[10]  T. Südhof,et al.  Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2‐domain? , 1998, The EMBO journal.

[11]  Y. Goda,et al.  Readily releasable pool size changes associated with long term depression. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. B. Rheuben,et al.  Transition from Growth Cone to Functional Motor Nerve Terminal inDrosophila Embryos , 1997, The Journal of Neuroscience.

[13]  E. Chapman,et al.  The C2 domains of synaptotagmin--partners in exocytosis. , 2004, Trends in biochemical sciences.

[14]  Kazuhiro Suzuki,et al.  Two Independent Pathways Mediated by cAMP and Protein Kinase A Enhance Spontaneous Transmitter Release atDrosophila Neuromuscular Junctions , 2000, The Journal of Neuroscience.

[15]  J. Buchanan,et al.  Morphologically Docked Synaptic Vesicles Are Reduced insynaptotagmin Mutants of Drosophila , 1998, The Journal of Neuroscience.

[16]  Ping Wang,et al.  The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis , 2001, The Journal of cell biology.

[17]  E. Chapman,et al.  Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca2+ , 2008, Nature Structural &Molecular Biology.

[18]  S. Benzer,et al.  Monoclonal antibodies against the Drosophila nervous system. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Littleton,et al.  Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse. , 1993, Development.

[20]  J. Littleton,et al.  Is synaptotagmin the calcium sensor? , 2003, Current Opinion in Neurobiology.

[21]  T. Südhof,et al.  Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C , 1990, Nature.

[22]  Hugo J. Bellen,et al.  Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca2+-activated neurotransmitter release , 1993, Cell.

[23]  M. Craxton.  Synaptotagmin gene content of the sequenced genomes , 2004, BMC Genomics.

[24]  Chun-Fang Wu,et al.  A MODIFIED MINIMAL HEMOLYMPH-LIKE SOLUTION, HL3.1, FOR PHYSIOLOGICAL RECORDINGS AT THE NEUROMUSCULAR JUNCTIONS OF NORMAL AND MUTANT DROSOPHILA LARVAE , 2004, Journal of neurogenetics.

[25]  T. Schwarz,et al.  Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain , 2002, Nature.

[26]  I. Robinson,et al.  The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo , 2002, Nature.

[27]  T. Südhof,et al.  Synaptotagmin-2 Is Essential for Survival and Contributes to Ca2+ Triggering of Neurotransmitter Release in Central and Neuromuscular Synapses , 2006, The Journal of Neuroscience.

[28]  Edwin R Chapman,et al.  How does synaptotagmin trigger neurotransmitter release? , 2008, Annual review of biochemistry.

[29]  J. Littleton,et al.  The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis , 2000, The Journal of cell biology.

[30]  Edwin R. Chapman,et al.  Synaptotagmin-Mediated Bending of the Target Membrane Is a Critical Step in Ca2+-Regulated Fusion , 2009, Cell.

[31]  W. Yamada,et al.  Time course of transmitter release calculated from simulations of a calcium diffusion model. , 1992, Biophysical journal.

[32]  T. Südhof,et al.  Membrane Fusion: Grappling with SNARE and SM Proteins , 2009, Science.

[33]  J. Littleton,et al.  Synaptotagmin I Functions as a Calcium Sensor to Synchronize Neurotransmitter Release , 2002, Neuron.

[34]  T. Südhof,et al.  A dual-Ca2+-sensor model for neurotransmitter release in a central synapse , 2007, Nature.

[35]  K. Broadie,et al.  Targeted Mutations in the Syntaxin H3 Domain Specifically Disrupt SNARE Complex Function in Synaptic Transmission , 2001, The Journal of Neuroscience.

[36]  Bill Adolfsen,et al.  Retrograde Signaling by Syt 4 Induces Presynaptic Release and Synapse-Specific Growth , 2005, Science.

[37]  Paul Tempst,et al.  SNAP receptors implicated in vesicle targeting and fusion , 1993, Nature.

[38]  Antonio Malgaroli,et al.  Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons , 1992, Nature.

[39]  V. Budnik,et al.  The drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure , 1994, Neuron.

[40]  T. Südhof,et al.  Differential but convergent functions of Ca2+ binding to synaptotagmin-1 C2 domains mediate neurotransmitter release , 2009, Proceedings of the National Academy of Sciences.

[41]  Zhiping P. Pang,et al.  Synaptotagmin-1 functions as the Ca2+-sensor for spontaneous release , 2009, Nature Neuroscience.

[42]  Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis. , 2007, Molecular biology of the cell.

[43]  T. Südhof,et al.  Three-Dimensional Structure of the Synaptotagmin 1 C2B-Domain Synaptotagmin 1 as a Phospholipid Binding Machine , 2001, Neuron.

[44]  T. Schwarz,et al.  The effect on synaptic physiology of synaptotagmin mutations in drosophila , 1994, Neuron.

[45]  G. Augustine,et al.  Dual Roles of the C2B Domain of Synaptotagmin I in Synchronizing Ca2+-Dependent Neurotransmitter Release , 2004, The Journal of Neuroscience.

[46]  C. Stevens,et al.  The Synaptotagmin C2A Domain Is Part of the Calcium Sensor Controlling Fast Synaptic Transmission , 2003, Neuron.

[47]  J. Littleton,et al.  A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth , 2007, Nature Neuroscience.

[48]  T. Südhof,et al.  Sr2+ Binding to the Ca2+ Binding Site of the Synaptotagmin 1 C2B Domain Triggers Fast Exocytosis without Stimulating SNARE Interactions , 2003, Neuron.

[49]  S. Sprang,et al.  Structure of the first C2 domain of synaptotagmin I: A novel Ca2+/phospholipid-binding fold , 1995, Cell.